Rheumatoid arthritis (RA) is a progressive destructive disorder that targets primarily the joints and is characterized by the hyperproliferation of synovial tissue and the infiltration of blood-derived cells resulting in the progressive erosion of the cartilage and bone. The incidence of RA has been reported to be around 30 per 100,000 population, and it may affect any age group from children to the elderly. The disease prevalence is about 1 percent worldwide. Thus, there are about 150,000 RA patients in the Netherlands only. The peak onset is between the ages of 30 and 55 and, because of the consistently higher rates in females, the prevalence of RA in females over 65 years is up to 5 percent.
RA is associated with a high degree of economic loss, morbidity, and early mortality. As an example, almost 80 percent of patients in one center were severely disabled after 20 years' follow-up; an additional one third had died. Patients with RA that require hospital care have at least a twofold increased mortality when compared to normals, and more severe RA is associated with higher mortality rates. The excess mortality in severe RA has been compared to that of three-vessel coronary artery disease or stage IV Hodgkin's disease.
An appreciation of the pathogenic mechanisms of RA and the poor outcomes with conventional therapy has led to the recent concept of aggressive treatment of newly diagnosed or early disease to suppress ongoing inflammation and prevent joint injury. Drug therapy is the mainstay of treatment for all patients except for those in clinical remission. Such therapy should be instituted with the goals of treating each patient sufficiently to induce a remission and preventing further loss of joint tissues or function in daily activities. In addition to conventional therapy with disease-modifying antirheumatic drugs, novel approaches aimed at TNF-α blockade have successfully entered the clinic. It is now possible to reach 20% improvement in about 70% of the RA patients using this approach. The majority of these American College of Rheumatology (ACR) 20% responders, however, will still have some actively inflamed joints. About 30% of the patients will not respond to TNF-α blockade with regard to arthritis activity.
Intra-articular corticosteroids are an important mainstay of the treatment of symptomatic synovitis in patients with RA. Especially when there is isolated arthritis activity under systemic antirheumatic therapy, as may occur in most patients, there is an indication for local treatment. However, not all patients will respond to the use of corticosteroids and its use is limited by side-effects.
The pathology of RA extends throughout the synovial joint. In contrast to the acellular nature of normal synovial fluid, RA synovial fluid is enriched predominantly with neutrophils, but macrophages, T-lymphocytes and dendritic cells are also present (Tak, P. P. Examination of the synovium and synovial fluid. In: Firestein G S, Panayi G S, Wollheim F A, editors. Rheumatoid arthritis. Frontiers in pathogenesis and treatment. New York: Oxford University Press, Inc., 2000: 55-68). The increase in cellularity is most obvious in the synovial membrane, which becomes infiltrated by cells recruited from the blood. The lining layer of the joint is increased from 1-2 cells to 6-8 cells thick and consists mainly of activated intimal macrophages and fibroblast-like synoviocytes. Alterations in the normal biology of synoviocytes are important in the development and maintenance of the pathologic process associated with RA, including invasion and destruction of articular cartilage and bone. In addition to the production of soluble mediators such as elastase and collagenase, synoviocytes mediate this pathophysiologic process by the expression of cell surface proteins, which are involved in the recruitment and activation of lymphocytes and macrophages within rheumatoid synovium. Synoviocytes are easily reached via the intra-articular space, are relatively long-lived, and thus represent an ideal target for gene therapy strategies (Chernajovsky, Y. et al., 1998, Drug Aging 12:29-41; Robbins, P. D. et al., 1998, Springer Semin. Immunopathol. 20:197-209).
In addition, the localized nature of the joint makes in vivo gene therapy very attractive. Many cellular and molecular interactions in the rheumatoid synovium are maintained and modulated by cytokines. A consistent finding in RA has been the abundance of fibroblast- and macrophage-derived proinflammatory cytokines such as IL-1, TNFα, and IL-18 in the rheumatoid synovium. The naturally occurring IL-1 and TNFα inhibitors, IL-1 receptor antagonist (IL-1RA) and the soluble TNFα receptors p55 and p75 are produced in parallel with their counterparts. For IL-18 an IL-18 binding protein is purified. Therapies providing excess recombinant cytokine inhibitors may shift the balance in RA towards an anti-inflammatory state. Clinical efficacy of anti-TNF-α and anti-IL-1 directed approaches emphasize that certain cytokines are appropriate targets for gene therapy. Another approach could be the directed overexpression of biologically active anti-inflammatory proteins (e.g. IL-4, IL-10, IL-13, and IFN-β) by synoviocytes to inhibit the inflammatory cascade (Boyle, D. L. et al., 1999, Gene Ther. 6:1911-1918).
NF-κB is clearly one of the most important regulators of pro-inflammatory gene expression (Tak, P. P. and Firestein, G. S., 2001, J. Clin. Invest. 107(1): 7-11). Synthesis of cytokines, such as TNF-α, IL-1β, IL-6, and IL-8 is mediated by NF-κB, as is the expression of Cox-2. Aupperle et al. (1999, J. Immunol. 163: 427-433) recently studied the role of IKK in primary fibroblast-like synoviocytes isolated from synovium of patients with RA and osteoarthritis. In both groups, immunoreactive IKK protein is abundant in these cells, and IKK-α and IKK-β are constitutively expressed at the mRNA level. IKK function in these cells can be greatly enhanced by TNF-α and IL-1, leading to degradation of endogenous IκB-α and nuclear translocation of NF-κB. Activation of this pathway and the consequent induction of IL-6, IL-8, ICAM-1, and collagenase-1 expression, depends specifically on IKK-β (Aupperle, K. R. et al., 1999, J. Immunol. 163: 427-433). Thus, transfection with adenoviral constructs encoding an IKK-β dominant negative mutant prevents TNF-α-mediated NF-κB nuclear translocation and pro-inflammatory gene expression in synoviocytes, whereas dominant negative IKK-α mutant has no effect (Aupperle, K. R. et al., 1999, J. Immunol. 163: 427-433).
Animal models of inflammatory arthritis support the notion that NF-κB activation plays a pathogenic role in vivo. For instance, increased synovial NF-κB binding precedes the development of clinical joint involvement in murine collagen-induced arthritis and gradually increases during the evolution of disease (Han, Z. N., et al. 1998, Autoimmunity 28: 197-208). Much of this binding activity appears to be due to p50, which has been implicated in collagenase-3 transcription and could contribute, along with locally activated AP-1, to extracellular matrix resorption. Synovial NF-κB activation also occurs within a few days after immunization in rat adjuvant arthritis (Tsao, P. W. et al. 1997, Clin. Immunol. Immunopathol. 83: 173-178). Selective activation of NF-κB in normal rats by intra-articular transfer of a functional IKK-β gene, leads to synovial inflammation and clinical signs of arthritis (Tak, P. P. et al., 2001, Arthritis Rheum. 44(8): 1897-907). Conversely, reduction of NF-κB nuclear translocation and clinical synovitis was observed in adjuvant arthritis in rats after an intra-articular injection with a dominant negative adenoviral IKK-β construct (Tak, P. P. et al., 2001, Arthritis Rheum. 44(8): 1897-907). The central role of NF-κB in inflammation has also been shown in rats with streptococcal cell wall-induced arthritis (Miagkov, A. V. et al., 1998, Proc. Natl. Acad. Sci. U.S.A. 95: 13859-13864) and in mice with collagen-induced arthritis (CIA) (Gerlag, D. M. et al., 2000, J. Immunol. 165: 1652-1658; Han, Z. N. et al. 1998, Autoimmunity 28:197-208).
Hence, various strategies aimed at increasing local production of anti-inflammatory proteins or aimed at inhibition of NF-κB activity in the synovial compartment by in vivo gene therapy hold great promise for the treatment of RA.
In order to enable sustained local production of effective doses of therapeutic proteins in the joint, in particular in the rheumatoid synovium, an efficient gene delivery system needs to be developed. A range of different viral and non-viral vectors exist, such as adenoviral vectors, adeno-associated virus vectors, retroviral vectors, herpes virus vectors, liposomes, DNA vaccination and the like (see Vervoordeldonk M. J. B. M and Tak P. P. 2001, Best Practice & Research Clinical Rheumatology Vol. 15 (5): 771-788). To date mainly adenoviral vectors have been tested as vectors for gene delivery. However, their episomal nature limits the duration of the gene expression, thereby making them not very suitable for the treatment of arthritis, where long-term gene expression is required.
Another disadvantage of adenoviral vectors is the presence of viral proteins, which may elicit an immune response in the host.
Adeno-associated viral vectors (AAV), on the other hand, have been shown (in some tissues) to integrate into the genome of the target cell (Hirata et al. 2000, J. of Virology 74:4612-4620), allowing long-term transgene expression in transduced cells. Adeno-associated virus is a helper-dependent DNA parvovirus, which is not associated with disease in humans or mammals (for review see Berns and Bohensky, 1987, Advances in Virus Research, Academic Press Inc, 32:243-307). Recombinant AAV vectors have been shown to be able to transfect a range of different cell types, such as hematopoietic cells, respiratory epithelial cells and neurons. However, for many cell types (such as for example synovial cells, but also many others) it remains unclear whether or not they can be transfected at all or efficiently by AAV vectors. Pan et al. (J. of Virology 1999, Vol 73, 4: 3410-3417) have been able to transfect rat synoviocytes showing symptoms of lipopolysaccharide induced arthritis using rAAV vectors, but they found that transgene expression diminished when inflammation subsided. Moreover, the literature reports widely divergent results from experiments attempting in vivo gene delivery to joints with AAV based vectors (Ghivizanni et al. 2000, Drug Discov. Today 6:259-267).
A complicating factor is that AAV serotypes differ in cellular tropism. WO99/61601 for example shows that AAV5 based vectors transduced certain cell types (cultured airway epithilial cells, cultured striated muscle cells and cultured human umbilical vein endothelial cells) at a higher efficiency than AAV2. On the other hand, AAV5 was much more inefficient in transducing cultured cos cells, 293, HeLa, IB3 cells and MCF7 cell lines, while both AAV2 and AAV5 showed poor transduction efficiencies for NIH 3T3, skbr3 and t-47D cell lines.
Despite the availability of the above viral and non-viral gene delivery systems, to date no suitable vector system exists for effective delivery of genes (encoding therapeutic proteins) to the rheumatoid synovium of subjects suffering from rheumatoid arthritis. There remains, therefore, a need to generate a suitable in vivo and ex vivo gene delivery system to the synovium in order to enable effective treatment. The present invention provides such a gene delivery system.